Osteoporosis is a systemic skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture. Although age-related bone loss occurs in both men and women, it begins earlier, and progresses more rapidly, in women. In the third or fourth decade of a woman's life, bone mass begins to decline because of an imbalance between the volume of mineral and matrix removed and that reincorporated during the bone remodeling process. When menopause occurs, the rate of bone loss accelerates and is particularly rapid in the first postmenopausal decade. This accelerated bone loss is caused by estrogen deficiency, which not only induces an enhanced, focal imbalance at remodeling sites but also increases the overall rate of remodeling. Riggs and Melton have written extensively on this topic, and have described two distinct syndromes of osteoporosis, type I and type II Riggs et al., N Engl J Med 314:1676-1686 (1986); Riggs et al., J Clin Endocrinol Metab 70:1229-1232 (1990)!. The former, type I, or "postmenopausal", osteoporosis, occurs in a relatively small subset of women 48-65 years of age and is linked to events related to menopause. The latter, type II, or "senile", osteoporosis, occurs in women greater than 75 years of age, and is the final result of gradual, age-related decline in bone mineral density. In this context, it is evident that women who experience osteoporotic bone fractures in the type I setting have lost considerable bone density compared to most other women of the same age. Thus, it is not surprising that some authors have written of "fast-losers" and "slow-losers" of bone density, distinguished from each other most particularly in the perimenopausal period of life. It is in this heterogeneity of osteoporosis that the potential role of estrogen and estrogen metabolism is greatest. All women undergo a dramatic decline in circulating estrogen levels with menopause, yet only 10-20% of this population ("fast-losers") will experience relatively rapid diminution of bone mineral density. Certain nutritional and lifestyle factors, such as inadequate intake of calcium, may contribute to low bone mass independent of estrogen level, and this can further increase a woman's risk of developing postmenopausal osteoporosis.
Postmenopausal osteoporosis affects the entire skeleton. In the early postmenopausal years, bone loss averages 2% per year but can vary from &lt;1% to &gt;5%. In the early phase of postmenopausal bone loss, the rate of trabecular (cancellous) bone loss exceeds that of cortical bone, and by the end of the first postmenopausal decade, most white women have osteopenia or osteoporosis. Postmenopausal osteoporosis is more prevalent in white and Asian women than in women of other races. It is estimated that osteoporosis affects about 45 percent of all postmenopausal white women.
Low bone mass is a major feature of postmenopausal osteoporosis and the primary determinant of fracture. The relationship between bone mass and fracture risk is more powerful than that between serum cholesterol concentration and coronary artery disease. Fortunately, bone mass can be readily measured, preserved, and even increased, with therapeutic intervention.
Fracture is the most clinically significant complication of postmenopausal osteoporosis. Hip fractures are costly and clinically serious. Among patients with hip fracture, 12 to 20% die within 1 year after the fracture, and more than 50% of the survivors are unable to return to independent living. Spinal injuries, in particular, may also lead to other adverse effects: loss of height, Kyphosis (Dowager's hump), and back pain (acute and chronic). Osteoporotic fractures often occur at a site of low bone mass and are usually induced by trauma.
Assessment of risk factors can help the physician to identify women who are susceptible to fracture, formulate a clinical suspicion of osteoporosis, and develop an osteoporosis prevention program. Several factors associ be altered by the patient. A knowledge of all existing risk factors, when considered in conjunction with the bone mineral density (BMD) measurement, may also provide direction for intervention with a specific therapeutic agent. A woman may be arbitrarily considered to be osteopenic or osteoporotic if she has a BMD greater than 1, or greater than 1.5 standard deviations below that of the typical woman of her age, respectively.
It is now possible, using advanced techniques, to determine appendicular and axial skeletal bone mass conveniently with a measurement accuracy exceeding 95 percent. Of the available methods of measuring bone mineral density (BMD), single-photon and single X-ray absorptiometry (SPA and SXA) are applicable to the peripheral appendicular bones, and dual-energy X-ray absorptiometry (DEXA) is the optimal method used to estimate axial, proximal appendicular, and total bone mass. Bone-mass measurement has an increasingly important role in clinical decision-making. Although factors such as body size, cigarette smoking, and reproductive history are valuable in determining which women are most likely to develop osteoporosis, it is useful to have reliable, quantitative evidence that a woman is at increased risk for fracture, especially when therapy is instituted. BMD measurement at any axial (that is, hip, vertebra) or peripheral (that is, radius, calcaneus) site is useful for a one-time assessment of fracture risk. Currently, however, AACE (American Association of Clinical Endocrinology) recommends performing the first measurement at the hip. The hip is also a good site for the baseline and follow-up measurements when therapeutic intervention is planned. Ideally, if resources allow, measurements should be taken at both sites for baseline and follow-up because the trabecular bone of the spine produces the quickest therapeutic response. These BMD measurements are often expressed as Z-scores, which represent the standard deviation of a woman's BMD from the norm; osteopenics have BMD Z-Scores &lt;-1, osteoporotics BMD Z Scores &lt;-1.5 (American Association of Clinical Endocrinology Guidelines). These cutoffs are somewhat arbitrary, given the high variability of BMD between women at any age.
A number of prospective studies using bone-mass measurements to predict fractures have been undertaken. Although each of the studies contains unique features, each has shown that decreased bone density at the sites measured is associated with increased risk of fracture at the sites reported.
Such studies demonstrate that bone-mass measurement, compared with the standard for age and weight, can predict which women can experience a fragility fracture. These groups concluded that bone mineral measurements taken at a variety of skeletal sites have a moderate ability to predict for at least eight to ten years a fracture that might be related to osteoporosis. Calculations vary with each study because of differences in populations, measurement techniques, etc.
From the studies shown, it appears that measurement of BMD has the greatest predictive value in determining future hip fracture, with relative risk ranging from 1.3 to 2.7. As an example, for every 1 SD decrease in BMD at any site, a 2.6-fold increase occurs in the risk of hip fracture. When adjusted for age, this means that a woman whose BMD is 1 SD below the mean is about seven times more likely to have a hip fracture than a woman whose bone density is 1 SD above the mean. In addition, there appears to be a trend in the three studies that measured BMD at the hip. Measurement of BMD at the hip is associated with the highest range of relative risks for subsequent hip fracture.
In summary, the lifetime risk of death from complications of hip fracture is about 2.8 percent for 50-year-old white postmenopausal women. Bone mineral density (BMD) is a good predictor of hip, spine, and all-site fractures. BMD measured at the proximal femur predicts subsequent risk of hip fracture better than BMD measured at other skeletal sites. A number of epidemiologic studies have eof BMD and the prevalence of fractures. The studies have determined that, regardless of the type of measurement used or the site measured, decreased BMD leads to increased risk of fracture.
The mechanism for loss of mineral density (BMD) in mammalian bone has been and continues to be the subject of intense research. One of the most active areas is elucidation of the role of estrogens in the formation and resorption of minerals in estrogen-sensitive tissue compartments of the bone. The clear cut role of estrogens in inducing and conservation of BMD, and preventing hip fracture after menopause Quigley et al. Am J Obstet Gynecol 156:1516-1523 (1987); Keil D et al. N Engl J Med 317:1169-1174 (1987)! has led to a long search for evidence of relative estrogen deficiency in women with osteoporosis. For example, Peak BMD is relatively low in women with primary amenorrhea or delayed puberty Dhuper et al., J Clin Endocrinol Metab 71: 1083-1088 (1990)!, and estrogen deficiency after menopause enhances the rate of bone turnover and results in an acceleration of bone loss Civitelli et al., J Clin Invest 82:1268-1274 (1988)!.
Studies of serum and urinary levels of estrogens in osteopenia and osteoporosis have, however, given negative, and often contradictory, results. Women lose bone at different rates up to 10 years after menopause, even though there is no significant differences in their total serum estradiol (E2) levels Riis Am J Med 98: (Suppl 2A)29S-32S (1995)!, or urinary estrone (E1) levels Lim et al., J Clin Endocrin Metab 82: 1001-1006 (1997)! as determined by immunoassay or gas chromatography-mass spectroscopy (GC-MS), respectively. Bone mineral density in premenopausal women, however, does seem to depend upon their total, lifetime exposure to circulating estrogen levels Civitelli et al. J Clin Invest 82:1268-1274 (1988)!. Moreover, the rate of bone loss in premenopausal women after complete oophorectomy is more rapid than in women undergoing natural menopause Stepan et al., Bone 8:279-284 (1987), and Civitelli et al., J Clin Invest 82:1268-1274 (1988)!. The levels of plasma E1 and E2 may not accurately reflect the biologically available amount of estrogen in postmenopausal women because these primary estrogens are further oxidized by intercellular microsomal enzymes in bone and other tissues to metabolites that have either more or less potent estrogenic effects in bone. For example, the inventor and others have recently shown by immunochemical methods that the metabolism of estrogen is altered in peri- and post-menopausal American women with osteopenia or osteoporosis such that production of urinary metabolites of estrogen, namely 2-hydroxyestrone (2OHE1) and/or 16.alpha.-hydroxyestrone (16.alpha.OHE1) are increased in osteopenia Hodge et al. J Bone Miner Res 10 (Suppl 1):S444 (1995)!. The later immunochemical studies of Hodge et al. found statistically significant negative correlations between BMD and urinary estrogen metabolites at several bone sites including spine and femur, but most especially between BMD and the lateral projections of the spine (VBD-LAT-tot). This initial finding, however, is in contrast to a recent study of urinary metabolites in Korean postmenopausal women by gas chromatography-mass spectrometry (GC-MS). Lim and colleagues J Clin Endocrinol Metab 82:1001-1006 (1997)! reported a positive significant correlation between 16.alpha.OHE1 and spinal BMD, and a possible negative correlation between 2-hydroxyestradiol (2OHE2) and femoral BMD. No correlations were found by Kim et al. for other urinary estrogen metabolites or between metabolite levels and BMD at other bone sites. The association between 2OHE2 and femoral bone density observed by Kim et al. was not significant, as correlation was lost after correcting for age. The later investigators did not specify which spinal projection(s) was used to calculate spinal BMD, nor did they examine the correlations between estrogen metabolites and BMD at other important sites such as the hip, or specific projections of hip BMD. The similarities and differences between these later two studies, however, should be interpreted cautiously in light of the established differences in drug metabolism and lifestyle between caucasians and orientals Lou, Drug Metab Rev 225:451-475 (1990)!.
Most significantly, the studies of Kim et al. (1997) or Hodge et al. (1995) were not designed to correlate serum or urinary estrogen metabolites with the rate of bone loss, or change in BMD with time in postmenopausal women. Although there is a correlation between BMD and risk for fracture, many women with low BMD at menopause lose BMD very slowly thereafter, and never experience fractures, even after trauma. Conversely, many women with high BMD at menopause will experience rapid bone loss and subsequent bone fracture.
Independent of BMD, the most important single diagnostic parameter for effective detection and management of osteopenic pathologies is the rate of bone loss. Current methods use x-ray densitometry or dual x-ray absorptiometry (DEXA) to assess BMD. Although the accuracy of DEXA methods are said to be as high as .+-.3 percent of the BMD, aortic calcification, vertebral compression, degenerative arthritis, or other spinal conditions and diseases, amongst other factors, add a higher error to measurement of BMD Nilas et al., Bone Miner 4:95-103 (1988)!. Moreover, changes in bone density over time are typically less than 5 percent/year in most postmenopausal osteopenics. Therefore, measurement intervals by DEXA for BMD of at least two years are needed before diagnostic confirmation of bone loss. Intermediate biomarkers of rate of bone loss, as in perimenopausal women, are needed to identify those women who can benefit from earlier treatment with estrogens, bisphosphonates, or other bone-conserving drugs. Although previous studies of serum and urinary estradiol and/or estrone in osteopenic women have yielded negative and contradictory results (see above), the dependence of BMD on estrogen suggests that some estrogen or its metabolite must be a marker for this physiological process. All women undergo a dramatic decline in circulating estrogen levels with menopause, yet only 10-20% of this population ("fast-losers) will experience relatively rapid diminution of bone mineral density. Preventive strategies depend upon identifying the clinical or biological characteristics of this small population, in order to target them for appropriate therapy. The levels of specific metabolites of estrone in physiological fluids from peri- and postmenopausal women will conceivably serve as such intermediate biomarkers.
The metabolism of estradiol, the ovarian estrogen, is primarily oxidative (FIG. 1). There is an initial oxidation of estradiol to estrone I, FIG. 1) which, in turn, is oxidized mainly by one of two alternative, irreversible pathways: 2-hydroxylation which leads to the relatively nonestrogenic metabolite 2-hydroxyestrone, and through activity of O-catechol methyl transferase (COMT), the inactive metabolite, 2-methoxyestrone (FIG. 1 , VIII and VII, respectively); and 16.alpha.-hydroxylation which leads to the estrogenic metabolite 16.alpha.-hydroxyestrone (III, FIG. 1), among others. The relative contribution of the 16.alpha.-hydroxylation pathway is relatively constant under most biologic circumstances. There are at least two other oxidative pathways for estrogen; 4-hydroxylation which leads to 4-hydroxyestrone (4OHE1)(IX), and 15.alpha.-hydroxylation which leads to 15.alpha.-hydroxyestrone (15OHE1) (X). Alterations may also exist in conjugation of estrogens in tissues and body fluids. Research done on urinary estrogen metabolites indicates that urinary estrogens may be covalently conjugated as ethers or esters with glucuronic acids, and/or sulphates, respectively, at the steroidal hydroxyl groups. Much less is known about the nature of estrogen metabolites in tissues and other bodily fluids. Hypothetical conjugates of 16OHE1 as they might occur in tissues and/or body fluids are illustrated in FIG. 2, conjugates of 2OHE1 in FIG. 3, and conjugates of 2MeoE1 in FIG. 4.
In 1966, Zumoff and associates reported that men with breast cancer demonstrated markedly increased 16.alpha.-hydroxylation of estradiol Zumoff et al. J Clin Endocrinol Metab 26: 960 (1966)!. These same investigators subsequently reported increased formation and excretion of urinary 16.alpha.-hydroxylated estrogen metabolites in women with breast cancer after injection of radiolabelled estradiol Hellman et al. J Clin Endocrinol Metab 33:138-144 (1971)!. Using this radiometric method, Fishman and coworkers greatly extended the study of 16.alpha.-hydroxylation and 2-hydroxylation in breast cancer by reporting the following findings: 1) increased 16.alpha.-hydroxylation, but unchanged 2-hydroxylation of estrogen was confirmed in women with breast cancer Schneider et al.: Proc Natl Acad Sci USA 79:3047-3051 (1982)!; 2) increased 16.alpha.-hydroxylation was found in women at familial high risk for breast cancer Bradlow et al.: Ann NY Acad Sci 464:138-151 (1986)!; and 3) Increased 16.alpha.-hydroxylation was found in mouse strains with high incidence of breast cancer, and the degree of increased risk paralleled the increase in 16.alpha.-hydroxylation Bradlow et al.: Proc Natl Acad Sci USA 82:6295-6299 (1985). By contrast, the aforecited radiometric studies found no significant alteration in 2-hydroxylation in breast cancer. No studies concerning bone metabolism or osteoporosis have been done using the radiometric method.
The radiometric method is, however, not applicable to routine medical practice, and is complicated by the necessity to normalize the amount of tritium released to the injected animal's body volume. Moreover, no information as to the amounts or kinds of estrogens transformed by 16.alpha.-hydroxylation of estradiol is obtained by a radiometric method. Recognizing the limitation of the radiometric method, Fishman and co-investigators attempted to develop a radioimmunoassay (RIA) for unconjugated 16OHE1 using polyclonal antisera to 16OHE1 and tritiated 16OHE1 as tracer Ikegawa et al., J Steroid Biochem 18:329-332 (1983)!. RIAs done upon ethyl ether extracts of serum found very low levels of 16OHE1 in serum from normal men and women, averaging only 4-10 pg/mL in men and 5-16 pg/mL in women. Unfortunately, the researchers found that blank values for water, buffer, and steroid-free serum were also in the range of 5-18 pg/mL. The very low levels of 16OHE1 found in this assay, and its lack of reproducibility, obviously precluded its use in further studies of the role of 16.alpha.-hydroxylation in breast cancer, and there are no reports of its use in any further studies. Yoshizawa and Fishman, J Clin Endocr 32:3-6 (1971), also attempted to develop an RIA for unconjugated 2OHE1 in methylene chloride extracts of serum. The later RIA found significant differences between clinical groups studied, but this assay was never used in studies of animals with cancer or other proliferative diseases. Emons and coworkers, in Acta Endocr, Copenh. 91: 158-166 (1979), developed an indirect radioimmunoassay for 2-methoxyestrone in human plasma, but did not apply that assay to studies of estrogen metabolism in disease. These same immunoassays were subsequently used by these investigators, however, in studies of estrogen metabolites in urine.
These assays also used the radioimmunoassay method and determined total urinary 2OHE1 and 16OHE1 (normalized to urine creatinine concentration) after deconjugation of glucuronides and sulfates with enzyme treatment. For example, Michnovicz et al. in Steroids 52:69-83, 1988, found no significant difference in total urinary 16OHE1 secretion when comparing smokers and nonsmokers, but increased 2-hydroxylation in smokers; Galbraith and Michnovicz in N. Engl. J. Med 321:269-274, 1989, found no effect of cimetidine on urinary total 16OHE1 secretion, but reported a decrease in 2-hydroxylation; and, Michnovicz and Galbraith in Steroids 55:22-26, 1990, found no effect of thyroxine treatment on total 16OHE1 secreted in urine, but increased 2-hydroxylation with thyroxine treatment.
Finding no differences in urinary secretion of an individual urinary metabolite reflective of suspected alterations in estrogen metabolism associated with a specific pathologic condition, Fishman and coworkers developed a method for detecting alterations in estrogen metabolism which comprised isolating at least two metabolites of estrone from a biological sample and determining their quotient. This quotient, and/or changes in this quotient, are reported to be reflective of alterations in estrogen metabolism. This method forms the basis of European Patent Application No. 040917682 published Jan. 23, 1991. In regard to the utility of measurements of 16OHE1, the inventors state in this Application (page 2, line 24-25) that, ". . . the constitutive nature of this metabolite has discouraged its further consideration for either diagnostic or therapeutic purposes". Moreover, the method of Michnovicz et al. does not recognize the importance of measuring the said glucuronide fraction of the estrogen metabolites, that is, the conjugated forms of metabolites, and specifically, the 3-glucuronides.
As indicated above, several methods have been used to detect altered estrogen metabolism, especially increased 16.alpha.-hydroxylation in animals bearing tumors. These methods, however, have not been applied to research in osteoporosis, and are not applicable to human research and routine medical practice. Previous attempts to quantify 16OHE1 directly by RIA have failed to find measurable levels in serum. Therefore, there exists a need in the medical art for rapid, accurate, diagnostic tests for metabolites of estrone such as 2OHE1, 2MeoE1, and 16OHE1, assays that reflect altered metabolism and conjugation of estrogens in tissues and bodily fluids from animals. The invention disclosed herein meets said need by providing for non-invasive diagnostic/prognostic assays to detect and/or quantify in mammalian tissues and body fluids, preferably urine and plasma or serum, estrogen metabolites 2OHE1, 2MeoE1, and 16OHE1 and their conjugates, preferably as the sum of the free metabolite and its 3-glucuronide conjugate.